High throughput quantification of the functional genes associated with RDX biodegradation using the SmartChip real-time PCR system.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a contaminant at many military sites. RDX bioremediation as a clean-up approach has been gaining popularity because of cost benefits compared to other methods. RDX biodegradation has primarily been linked to six functional genes (diaA, nfsI, pnrB, xenA, xenB, xplA). However, current methods for gene quantification have the risk of false negative results because of low theoretical primer coverage. To address this, the current study designed new primer sets using the EcoFunPrimer tool based on sequences collected by the Functional Gene Pipeline and Repository and these were verified based on residues and motifs. The primers were also designed to be compatible with the SmartChip Real-Time PCR system, a massively parallel singleplex PCR platform (high throughput qPCR), that enables quantitative gene analysis using 5,184 simultaneous reactions on a single chip with low volumes of reagents. This allows multiple genes and/or multiple primer sets for a single gene to be used with multiple samples. Following primer design, the six genes were quantified in RDX-contaminated groundwater (before and after biostimulation), RDX-contaminated sediment, and uncontaminated samples. The final 49 newly designed primer sets improved upon the theoretical coverage of published primer sets, and this corresponded to more detections in the environmental samples. All genes, except diaA, were detected in the environmental samples, with xenA and xenB being the most predominant. In the sediment samples, nfsI was the only gene detected. The new approach provides a more comprehensive tool for understanding RDX biodegradation potential at contaminated sites.

The Ribosomal Database Project: improved alignments and new tools for rRNA analysis.

The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/.

The Ortega Hypothesis: Citation analysis suggests that only a few scientists contribute to scientific progress.

Let us consider, then, some general conclusions that may be drawn from the findings reported in this study. The data allow us to question the view stated by Ortega, Florey, and others that large numbers of average scientists contribute substantially to the advance of science through their research. It seems, rather, that a relatively small number of physicists produce work that becomes the base for future discoveries in physics. We have found that even papers of relatively minor significance have used to a disproportionate degree the work of the eminent scientists. Although the conclusions of this paper may be reasonably clear, the implications of these data for the structure of scientific activity, at least in physics, need careful consideration.

Chance and consensus in peer review.

An experiment in which 150 proposals submitted to the National Science Foundation were evaluated independently by a new set of reviewers indicates that getting a research grant depends to a significant extent on chance. The degree of disagreement within the population of eligible reviewers is such that whether or not a proposal is funded depends in a large proportion of cases upon which reviewers happen to be selected for it. No evidence of systematic bias in the selection of NSF reviewers was found.

The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data.

Substantial new features have been implemented at the Ribosomal Database Project in response to the increased importance of high-throughput rRNA sequence analysis in microbial ecology and related disciplines. The most important changes include quality analysis, including chimera detection, for all available rRNA sequences and the introduction of myRDP Space, a new web component designed to help researchers place their own data in context with the RDP's data. In addition, new video tutorials describe how to use RDP features. Details about RDP data and analytical functions can be found at the RDP-II website (http://rdp.cme.msu.edu/).

The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis.

The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic framework for these data. Updated monthly, these services are made available through the RDP-II website (http://rdp.cme.msu.edu/). RDP-II release 9.21 (August 2004) contains 101,632 bacterial small subunit rRNA gene sequences in aligned and annotated format. High-throughput tools for initial taxonomic placement, identification of related sequences, probe and primer testing, data navigation and subalignment download are provided. The RDP-II email address for questions or comments is rdpstaff@msu.edu.

rrndb: the Ribosomal RNA Operon Copy Number Database.

The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes, information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections. Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme. msu.edu.

The RDP-II (Ribosomal Database Project).

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [Nucleic Acids Res. (2000), 28, 173-174], continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 8.0 (June 1, 2000) consisted of 16 277 aligned prokaryotic small subunit (SSU) rRNA sequences while the number of eukaryotic and mitochondrial SSU rRNA sequences in aligned form remained at 2055 and 1503, respectively. The number of prokaryotic SSU rRNA sequences more than doubled from the previous release 14 months earlier, and approximately 75% are longer than 899 bp. An RDP-II mirror site in Japan is now available (http://wdcm.nig.ac.jp/RDP/html/index.h tml). RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment polymorphism experiments. The RDP-II email address for questions and comments has been changed from curator@cme.msu.edu to rdpstaff@msu.edu.

The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy.

The Ribosomal Database Project-II (RDP-II) pro-vides data, tools and services related to ribosomal RNA sequences to the research community. Through its website (http://rdp.cme.msu.edu), RDP-II offers aligned and annotated rRNA sequence data, analysis services, and phylogenetic inferences (trees) derived from these data. RDP-II release 8.1 contains 16 277 prokaryotic, 5201 eukaryotic, and 1503 mitochondrial small subunit rRNA sequences in aligned and annotated format. The current public beta release of 9.0 debuts a new regularly updated alignment of over 50 000 annotated (eu)bacterial sequences. New analysis services include a sequence search and selection tool (Hierarchy Browser) and a phylogenetic tree building and visualization tool (Phylip Interface). A new interactive tutorial guides users through the basics of rRNA sequence analysis. Other services include probe checking, phylogenetic placement of user sequences, screening of users' sequences for chimeric rRNA sequences, automated alignment, production of similarity matrices, and services to plan and analyze terminal restriction fragment polymorphism (T-RFLP) experiments. The RDP-II email address for questions or comments is rdpstaff@msu.edu.

Changes in the half-life of ribosomal protein messenger RNA caused by translational repression.

The half-life of ribosomal protein operon L11 mRNA in vivo was measured during exponential growth by following the kinetics of incorporation of radioactive precursors into L11 mRNA transcribed from multi-copy plasmids. The degree of translational feedback regulation by L1, the L11 operon-specific translational repressor protein, was changed by altering the site on the "L11 mRNA" where L1 interacts. The half-life of the overproduced L11 mRNA increased by about fivefold when translational repression was abolished, while the half-life of mRNA from the spc ribosomal protein operon, which is not translationally regulated by L1, stayed constant. Furthermore, the half-life of L11 operon mRNA carrying an additional mutation in the ribosome binding site that abolishes translation remains short. This indicates that the change in half-life observed during increased gene dosage is due to translational repression by L1 and is probably a consequence of L1 blocking translation of L11 mRNA and not due to some nucleolytic activity mediated by L1.

Feedback regulation of rRNA synthesis in Escherichia coli. Requirement for initiation factor IF2.

It has been shown that the transcription of rRNA in Escherichia coli is feedback-regulated by its own transcription products through a negative feedback loop which appears to require the assembly of rRNA into complete ribosomes. In order to examine whether the feedback loop involves the ribosomes' main function, translation, we have constructed a strain in which the chromosomal copy of infB, encoding IF2, was placed under lac promoter/operator control, and the effects of limitation of translation initiation factor IF2 on the regulation were examined. By varying the concentration of a lac operon inducer, isopropyl thiogalactoside (IPTG), it was possible to vary the cellular concentration of IF2. Under the growth conditions used, decreasing the concentration of IF2 about twofold affected the growth rate only slightly, but further deprivation of IF2 resulted in a significant decrease in growth rate, an increase in RNA content and a large accumulation of non-translating ribosomes. These accumulated ribosomes were apparently unable to cause feedback regulation of rRNA synthesis in the absence of sufficient IF2. When a higher concentration of IPTG was added to these IF2-deficient cells, a rapid increase in the IF2 level and a significant decrease in the rate of RNA accumulation were observed before the new steady-state growth was attained. These results indicate that IF2 apparently is necessary for feedback regulation of stable RNA and imply that ribosomes must enter translation for feedback regulation to occur.

Supplementation of monensin and Optimase to beef cows consuming low-quality forage during late gestation and early lactation.

Two experiments were designed to investigate the effects of feeding monensin and/or slow release urea with a fibrolytic feed enzyme (Optimase; Alltech, Inc., Nicholasville, KY) on performance, milk production, calf growth performance, and blood metabolites in beef cows. Spring-calving cows and heifers were used in a completely randomized design in Exp. 1 (N = 84; 534 ± 68 kg initial BW) and Exp. 2 (N = 107; 508 ± 72 kg initial BW). Exp. 1 supplements were formulated to meet cow protein requirements and fed daily and included 1) cottonseed meal with no monensin (control); or 2) monensin added to control to supply 200 mg per head per d (MON). In Exp. 2, experimental supplements included 1) cottonseed meal/wheat middlings (CS) fed at a rate to provide adequate DIP and CP according to , 2) the CS plus soybean hulls and 61 g per cow per d Optimase (OPT), 3) the CS plus monensin to supply 200 mg per cow per d (MON2), and 4) OPT plus MON2 (Combo). Cows were fed in last trimester through early lactation in Exp. 1 and during 2nd trimester in Exp. 2. Data were analyzed using the Mixed procedure in SAS with animal as the experimental unit. In Exp. 1, treatment did not affect cow BW or BCS change (P > 0.19). Calf birth BW was not affected by dam treatment (P = 0.24); however, calves from dams consuming MON weighed more (P < 0.04) at d 45 and at trial end. Calves also had greater (P = 0.04) ADG from birth to trial end. Milk production did not significantly differ among treatments (P > 0.41). In Exp. 2, mean cow BW and BCS were similar (P > 0.35) among treatments on d 90. However, from d 0 to 54, cows assigned to the OPT supplement gained less BCS (P = 0.02) compared with cows assigned to the CS supplement. Cumulative BCS gain was greater (P < 0.01) for CS-fed cows than for cows fed the OPT and MON2 supplements, although it was not significantly different for cows fed the Combo supplement. These studies indicate that the influence of monensin on cow BW and BCS change is inconsistent. The potential for monensin supplementation to positively impact calf performance during early lactation seems to be clearer. Replacing a portion of oilseed N in the supplement with Optimase may marginally reduce cow performance. Further research is needed to determine both the effects of monensin and the implications of combining monensin with Optimase on forage intake and cow performance at various stages of production.

Characterization of human liver thermostable phenol sulfotransferase (SULT1A1) allozymes with 3,3',5-triiodothyronine as the substrate.

Sulfotransferase 1A1 (SULT1A1) (thermostable phenol sulfotransferase, TS PST1, P-PST) is important in the metabolism of thyroid hormones. SULT1A1 isolated from human platelets displays wide individual variations not only in the levels of activity, but also in thermal stability. The activity of the allelic variant or allozyme SULT1A1*1, which possesses an arginine at amino acid position 213 (Arg213) has been shown to be more thermostable than the activity of the SULT1A1*2 allozyme which possesses a histidine at this position (His213) when using p-nitrophenol as the substrate. We isolated a SULT1A1*1 cDNA from a human liver cDNA library and expressed both SULT1A1*1 and SULT1A1*2 in eukaryotic cells. The allozymes were assayed using iodothyronines as the substrates and their biochemical properties were compared. SULT1A1*1 activity was more thermostable and more sensitive to NaCl than was SULT1A1*2 activity when assayed with 3,5,3'-triiodothyronine (T(3)). Sensitivities to 2,6-dichloro-4-nitrophenol (DCNP) and apparent K(m) values for SULT1A1*1 and for SULT1A1*2 with iodothyronines were similar. Based on K(m) values, the preferences of these SULT1A1 allozymes for iodothyronine substrates were the same (3,3'-diiodothyronine (3,3'-T(2))>3', 5',3-triiodothyronine (rT(3))>T(3)>thyroxine (T(4))>>3,5-diiodothyronine (3,5-T(2))). SULT1A1*1 activity was significantly higher than the SULT1A1*2 activity with T(3) as the substrate. Potential differences in thyroid hormone sulfation between individuals with predominant SULT1A1*1 versus SULT1A1*2 allozymes are most likely due to differences in catalytic activity rather than substrate specificity.

Impairment of the asialoglycoprotein receptor by ethanol oxidation.

It is well established that ethanol exposure impairs the process of receptor-mediated endocytosis in hepatic cells, although the molecular mechanism(s) and the physiological consequence(s) of this impairment are unclear. Because addressing these mechanistic questions is difficult in vivo, we have developed a recombinant cell line of hepatic origin capable of metabolizing ethanol. In this study, we have used these recombinant cells, designated HAD cells, to investigate the ethanol-induced impairment to the receptor-mediated endocytosis of the hepatic asialoglycoprotein receptor. Comparing the binding of the ligand asialoorosomucoid in both the parental Hep G2 cells and the recombinant HAD cells, maintained in the presence and absence of ethanol, revealed decreased ligand binding in the HAD cells. This impairment was accentuated by prolonging the ethanol exposure, reaching approximately 40% in both surface and total receptor populations by 7 days. Addition of the alcohol dehydrogenase inhibitor pyrazole to the ethanol-containing medium abolished this impairment, indicating that the decreased binding was a result of the alcohol dehydrogenase-mediated oxidation of ethanol. Furthermore, using antibody specific to the asialoglycoprotein receptor, it was demonstrated that the ethanol-induced impairment in ligand binding was a consequence of decreased ligand binding and not a result of diminished receptor numbers. These results indicated that ethanol oxidation was required for the ethanol-induced impairment in ligand binding, and that the reduced ligand binding was a result of a decrease in the ability of the ligand to bind to the receptor.

Symptomatic stage C carcinoma of prostate. Traditional therapy.

Of 119 consecutive patients with adenocarcinoma of the prostate, 54 patients had clinical Stage C disease and are the subject of this study. Of these 54 patients, 52 underwent transurethral prostatectomy and bilateral orchiectomy as their initial treatment; estrogens were not prescribed. The mean age of the patients with Stage C disease was seventy-three years, the average survival 6.4 years, and the five and ten-year survivals were 66 and 20 per cent, respectively. Stage C patients treated in this series did as well as those treated with radiation or radical extirpation.

Effects of macrophage inhibitory factor-A3 (MIF-A3) on cytokine secretion and phagolysosome fusion in murine macrophages.

Macrophage inhibitory factor-A3 (MIF-A3) is a fraction derived from Mycobacterium avium serovar 2 (Mav2) that consists of a small amine containing compound (peptide), trehalose and two or three short chain fatty acids. MIF-A3 has been shown to inhibit candidacidal activity of murine thioglycolate-elicited peritoneal-derived macrophages and bovine peripheral blood monocytes, and scavenge reactive oxygen intermediates. In this study, MIF-A3 was evaluated for its effect on secretion of IL-1beta, IL-6, IL-10, TNFalpha and GM-CSF in C57BL/6 murine thioglycolate-elicited peritoneal-derived macrophages, with and without pre-incubation with affinity purified goat anti-MIF-A3 IgG, using ELISA cytokine kit analysis. Results of this study suggest that anti-MIF-A3 IgG does not enhance clearance of Mav2, alter phagocytosis or alter phagosome-lysosome interactions as determined by electron microscopy in Mav2 infected macrophages. MIF-A3 does induce secretion of IL-6, but does not induce secretion of TNFalpha, IL-1beta, and GM-CSF. TNFalpha has been previously shown to reduce growth, while IL-6 has been shown to enhance growth of M. avium. Since IL-6 appears to enhance growth of M. avium and MIF-A3 induces IL-6 secretion, MIF-A3 may be responsible for enhanced intracellular growth in M. avium infections and be a factor in the pathogenesis of M. avium infections.

Efficacy of vaccination for Mycobacterium avium with whole cell and subunit vaccines in experimentally infected swine.

Mycobacterium avium infections are a common problem in large swine producing states and cause substantial financial losses at slaughter inspection due to carcass condemnation. Once the infection is established in a swine herd it is difficult to effectively prevent or eliminate the disease. Previous mouse studies in our laboratory suggested that Macrophage Inhibitory Factor-A3 (MIF-A3) is a virulence factor of M. avium and potential antigen for vaccine development. In this study we evaluated the efficacy of a killed 'whole cell' M. avium serovar 2 bacterin and conjugated MIF-A3 subunit vaccine in preventing infection and disease in swine challenged with virulent M. avium serovar 2. Gross and microscopic pathology, acid-fast staining, culture and polymerase chain reaction (PCR) for the M. avium specific insertion sequence IS902 were utilized in evaluation. Results indicated that neither vaccine prevented infection in challenged animals; however, a 47% reduction in severity of disease was found in swine vaccinated with the 'whole cell' M. avium serovar 2 bacterin. Reduction in severity of disease was not detected in animals vaccinated with the subunit MIF-A3 vaccine.